Necroptosis Stimulates Interferon-Mediated Protective Anti-Tumor Immunity

Necroptosis is an inflammatory form of cell suicide that critically depends on the kinase activity of Receptor Interacting Protein Kinase 3 (RIPK3). Previous studies showed that immunization with necroptotic cells conferred protection against subsequent tumor challenge. Since RIPK3 can also promote apoptosis and NF-κB-dependent inflammation, it remains difficult to determine the contribution of necroptosis-associated release of damage-associated molecular patterns (DAMPs) in anti-tumor immunity. Here, we describe a system that allows us to selectively induce RIPK3-dependent necroptosis or apoptosis with minimal NF-κB-dependent inflammatory cytokine expression. In a syngeneic tumor challenge model, immunization with necroptotic cells conferred superior protection against subsequent tumor challenge. Surprisingly, this protective effect required CD4+ T cells rather than CD8+ T cells and is dependent on host type I interferon signaling. Our results provide evidence that death-dependent type I interferon production following necroptosis is sufficient to elicit protective anti-tumor immunity.


Introduction
Necroptosis is widely viewed as an in ammatory form of cell death due to the release of DAMPs from ruptured plasma membrane.Receptor interacting protein kinase 3 (RIPK3), the essential serine/threonine kinase in necroptosis, can be activated by one of three distinct upstream activators: the related kinase RIPK1, the toll-like receptor 3 (TLR3) and TLR4 adaptor TIR domain containing adaptor molecule 1 (TRIF), and the viral RNA sensor Z-DNA binding protein 1 (ZBP1).RIPK3 oligomerizes and forms cytosolic signaling complexes via its RIP homotypic interaction motif (RHIM), which allows it to phosphorylate the necroptosis effector molecule Mixed Lineage Kinase domain-Like (MLKL).Phosphorylation causes MLKL to oligomerize and translocate to the plasma membrane to induce membrane rupture, leading to the leakage of damage associated molecular patterns (DAMPs) and in ammatory responses (1,2).In addition to promoting necroptosis, RIPK3 can also participate in apoptosis in certain situations.For instance, in the absence of MLKL or when the kinase activity of RIPK3 is inhibited, RIPK3 can stimulate formation of an alternate apoptosis-inducing complex with caspase-8, RIPK1, and FAS-associated death domain protein (FADD) (3).Moreover, RIPK3 can also stimulate in ammatory gene expression in a RHIMdependent but cell death-independent manner (4)(5)(6)(7).In this context, the RHIM serves as a scaffold to stimulate NF-κB activation (4,8,9).RIPK3 expression is downregulated in many tumor types, suggesting that RIPK3 has important functions in tumor suppression (10,11).In support of its anti-tumor role, lower RIPK3 expression correlates with worsened patient survival in lung cancer (12), chronic lymphocytic leukemia (13), colon cancer (14), malignant mesothelioma (15), and breast cancer (16).In contrast, tumor RIPK3 expression appears to be bene cial for tumor immune surveillance.For instance, expression of RIPK3 and other necroptotic adaptors in tumor cells was associated with improvement in CD8 + T cell in ltration in hepatocellular carcinoma (17), cholangiocarcinoma (18), and prostate cancer (19).
The current gold standard approach for studying the immunogenicity of cell death in vivo is to use dead or dying cells as a tumor vaccine to immunize syngeneic mice (20).Previous studies have demonstrated that RIPK3-induced necroptosis promotes dendritic cell (DC) cross-priming of tumor-speci c CD8 + T cells to achieve control of tumor growth.However, necroptosis was accompanied by strong NF-κB-dependent cytokine expression in these studies.Thus, it was not possible to distinguish the direct contribution of necroptosis-induced DAMPs release in the induction of anti-tumor immunity (21)(22)(23)(24).Further, cellintrinsic NF-κB signaling during necroptosis can also promote carcinogenesis (25).Consequently, there remains a need to clarify the consequences of these distinct aspects of tumor RIPK3 signaling in the antitumor response.
We previously showed that in a doxycycline (DOX)-inducible system, RIPK3 expression accompanied by proteasome inhibition was su cient to drive necroptosis in 3T3 broblasts.Here, we adopted this DOXinducible system in tumor cells.In contrast to other models in which RIPK3 activation was achieved using chemical-induced dimerization of synthetic RIPK3 chimeric cassettes (3), RIPK3 activation in our system was independent of RIPK1 and did not induce strong NFκB activation.By selectively restricting cell death to either RIPK3-dependent apoptosis or necroptosis, we found that immunization with necroptotic cells, but not apoptotic cells showed marked protection to subsequent tumor challenge.Surprisingly, immunization with necroptotic cells stimulated an anti-tumor CD4 + T cell response while CD8 + T cells were dispensable for tumor protection.The protection conferred by necroptotic cell immunization was observed with tumors from different tissue origin.Mechanistically, we showed that interferon beta (IFNβ) was speci cally induced in necroptosis but not in apoptosis, and blocking cell death effectively eliminated this type I IFN response.Furthermore, the protective effect of necroptosis immunization was abrogated when host IFN signaling was inhibited by IFNAR de ciency.These data suggest that necroptosis in the absence of NF-κB dependent cytokine expression drives anti-tumor immunity through a distinct type I IFN and CD4 + T cell dependent mechanism.

Expression of RIPK3 accompanied by proteasome inhibition drives tumor cell necroptosis
RIPK3 expression is frequently inhibited in tumor cells through promoter hypermethylation (26).To explore whether re-expression of RIPK3 might enhance anti-tumor immune surveillance, we rst attempted to restore expression of endogenous RIPK3 in Lewis Lung Carcinoma cells expressing chicken ovalbumin (LLC-OVA) using the DNA methyltransferase inhibitor 5-AZA-dC.Although 5-AZA-dC successfully restored RIPK3 expression, its toxicity prevented further exploration of cell death responses (Fig. 1a and data not shown).We therefore opted to utilize a doxycycline (DOX)-inducible system to restore RIPK3 expression in LLC-OVA cells.Consistent with previous report (3), DOX-induced expression of RIPK3 was not su cient to cause cell death of the LLC-OVA cells.However, addition of the proteasome inhibitor MG132 to prevent RIPK3 degradation led to strong cell death in DOX-induced LLC-OVA cells (Fig. 1b).This cell death was dependent on RIPK3 expression, since MG132 alone did not compromise cell survival.Increased phosphorylation of MLKL (pMLKL) was observed in DOX-and MG132-treated cells, indicating that necroptosis was the dominant form of cell death (Fig. 1c).The RIPK3 kinase inhibitor GSK'843 did not inhibit cell death (Fig. 1d), although it effectively inhibited pMLKL (Fig. 1e).Rather, GSK'843 increased caspase-3 cleavage (Fig. 1e), suggesting a switch from necroptosis to apoptosis (6).Indeed, co-treatment with GSK'843 and the pan-caspase inhibitor zVAD-fmk largely abrogated pMLKL, caspase-3 cleavage and cell death (Fig. 1d-e).Since pMLKL and caspase-3 cleavage required DOX-induced RIPK3 expression (Fig. 1e), these results indicate that RIPK3 has the capacity to promote necroptosis as well as apoptosis.

Selective induction of RIPK3-dependent tumor cell necroptosis or apoptosis
The switch from necroptosis to apoptosis with RIPK3 kinase inhibitor revealed a possible method to manipulate RIPK3-dependent cell death.To circumvent the off-target effects of chemical inhibitors, we utilized the CRISPR/Cas9 system to inactivate either caspase-8 (Casp8) or Mlkl in the DOX-inducible LLC-OVA cells (WT).Casp8-KO and MLKL-KO cells underwent DOX-and MG132-induced cell death with similar kinetics and magnitude when compared to WT cells (Fig. 1f).As in the case of RIPK3 kinase inhibition, MLKL-KO cells exhibited increased caspase-3 cleavage, indicating a switch from necroptosis to apoptosis (Fig. 1g).By contrast, Casp8-KO retained pMLKL but did not exhibit any caspase-3 cleavage (Fig. 1g).Knockout of both Mlkl and Casp8 completely abrogated cell death (Fig. 1h-i).Hence, selective activation of RIPK3-dependent necroptosis and apoptosis was achieved by genetic inactivation of Casp8 and Mlkl respectively (Fig. 1j).
Taken together, these data indicate that RIPK1-and NF-κB-dependent cytokine production was absent in our necroptosis induction system.

Immunization with necroptotic cells protects against tumor challenge
Immunogenic cell death (ICD) such as necroptosis was widely thought to stimulate immune responses through the release of DAMPs.We tested this premise using our LLC-OVA cell lines.Casp8-KO or MLKL-KO LLC-OVA cells were treated with DOX and MG132 to induce necroptosis and apoptosis respectively, and co-cultured with splenic DCs from Flt3L-treated mice.We found that necroptotic cells (NEC) bolstered expression of the costimulatory molecule CD80 on multiple DC subsets and monocytes compared to either apoptotic cells (APOP) or untreated controls (Fig. 3a-b, Supplemental Fig. 2), consistent with the notion that necroptosis is more immunogenic than apoptosis.
We next immunized mice with NEC or APOP subcutaneously followed by challenge with live LLC-OVA cells on the opposite ank eight days post-immunization (Fig. 3c).Importantly, when compared to PBS or APOP-immunized groups, tumor growth was signi cantly blunted by NEC immunization (Fig. 3d).NEC immunization similarly protected the hosts from subsequent challenge with B16-F1 melanoma (Fig. 3e).Hence, NEC immunization provides protection against tumors of different tissue origin.NEC immunization increased overall T cell in ltration in the tumor (Fig. 3f).In contrast to previous studies in which necroptosis was shown to be superior in promoting CD8 + T cell responses (21)(22)(23), effector/memory CD8 + T cell (CD8 + CD44 hi CD62L − ) in ltration was similar between NEC and APOP immunization groups (Fig. 3g).By contrast, overall CD4 + T cell and effector CD4 + CD44 hi CD62L − T cell in ltration was signi cantly elevated in NEC-immunized mice compared to APOP-immunized or PBStreated mice (Fig. 3h).

Necroptosis immunization stimulates CD4 + T celldependent anti-tumor immunity
Increase in CD8 + and CD4 + T cell in ltration in NEC-immunized mice in comparison to PBS control group was already evident on day 5 post-implantation when tumors were rst palpable (Fig. 4a-b).This is in contrast to tumor myeloid populations, which were comparable at this timepoint (Supplemental Fig. 3).
To test the contribution of CD4 + and CD8 + T cells in the protective effect conferred by NEC immunization, we used antibodies to deplete these populations.Importantly, only the depletion of CD4 + cells, but not CD8 + cells prior to immunization was able to abrogate the tumor protection by NEC immunization (Fig. 4c).Consistent with the dispensable role of CD8 + T cell in this process, disrupting cDC1-dependent cross-priming of CD8 + T cells using Batf3-de cient hosts did not affect the tumor suppressive effect of NEC immunization (Fig. 4d).In aggregate, these data suggest that NEC immunization confers anti-tumor restriction through CD4 + T cells and independent of CD8 + T cells.

Full CD4 + T cell priming requires secondary challenge with live tumor cells
To test whether CD4 + T cell priming occurs during the immunization phase, we adoptively transferred CD4 + T cells from OT-II mice and found that NEC immunization did not enhance OT-II CD4 + T cells accumulation in the draining lymph node (Supplemental Fig. 4a), even after a second immunization with NEC cells (Supplemental Fig. 4b-c).In contrast, increased CD4 + OT-II T cells accumulation in response to NEC immunization was detected in the draining lymph nodes (Fig. 5b-c) and tumor (Fig. 5d-f) when OT-II T cells were adoptively transferred the day prior to tumor challenge (Fig. 5a).This effect was evident whether the host received a single or multiple doses of NEC immunization (Supplemental Fig. 4d-e).

Type I interferon mediates protection conferred by necroptosis immunization
To interrogate the mechanism by which NEC immunization conferred protection against tumor challenge, we performed bulk RNA sequencing on the tumor tissues.We found that the gene expression largely clustered based on the different immunization regimen (Fig. 6a).In comparing the top differentially expressed genes between the NEC-and APOP-immunized groups, we found that the majority were interferon stimulated genes (ISGs) (Fig. 6b).Gene Set Enrichment Analysis (GSEA) further con rmed that NEC-immunized tumors showed enrichment for genes involved in response to interferon-beta (IFN-β) (Fig. 6c-d).Increased expression of several ISGs in tumors from NEC-immunized mice compared to those from APOP-immunized mice was further validated by qPCR (Fig. 6e).
We next sought to determine whether this IFN response might have originated from the dying tumor cells.
Strikingly, we detected production of IFN-β and ISGs in necroptotic cells, but not apoptotic or DKO cells that lack Casp8 and MLKL.(Fig. 7a).Moreover, NEC immunization in Ifnar1 −/− mice failed to improve tumor control (Fig. 7b).Furthermore, IFNAR neutralizing antibody also abrogated the NEC immunizationmediated protection (Fig. 7c).Consistent with the notion that CD4 + T cells are critical for NEC immunization-mediated tumor control, CD4 + T cell in ltration was reduced in Ifnar1 −/− mice compared to WT controls (Fig. 7d).By contrast, NEC-induced tumor suppression and CD4 + T cell in ltration was comparable between WT and Ifngr −/− mice (Supplemental Fig. 5).These data suggest that necroptosis stimulates cell-intrinsic IFN-β production to initiate a cascade of reaction that triggers host type I IFN signaling to bolster anti-tumor CD4 + T cell responses.

Discussion
RIPK3 signaling can stimulate necroptosis, apoptosis, and death-independent in ammatory cytokine production.To further complicate matters, DAMPs release from dying cells can also promote in ammatory gene expression.The di culty in separating these diverse signaling events has led to con icting reports on the role of RIPK3 signaling in anti-tumor immunity.Here, we utilized a system to drive RIPK3-dependent necroptosis or apoptosis without strong death-independent cytokine expression to interrogate the impact of necroptosis-associated DAMPs release in tumor immunity.Using this system, we observed that prophylactic immunization with necroptotic cells was su cient to drive protective antitumor CD4 + T cell responses.Although both CD8 + and CD4 + T cell in ltration was enhanced, depletion experiments revealed that only CD4 + T cells were indispensable for this protective effect.The enhanced recruitment of CD4 + T cells is dependent on host type I interferon signaling, consistent with the wellknown role of interferon in anti-tumor response.
Our results differ from several previous studies in which chemical-induced dimerization of chimeric RIPK3 fusion proteins led to necroptosis and concomitant RIPK1-dependent cytokine expression (21)(22)(23).The lack of RIPK1 engagement and cytokine expression in our system might be due to the use of native RIPK3 rather than RIPK3 fusion cassettes, which causes a lower level of RIPK3 nucleation.Due to the natural turnover of RIPK3, proteasome inhibition is required to elicit cell death and to unleash full protective antitumor immune responses.Since our system does not require expression of a foreign dimerization cassette, it also avoids issues such as immune reaction against exogenously introduced protein antigens.
Our results differ from the traditional view that ICD mainly stimulates DC cross-priming of CD8 + T cells to promote tumor protection (27,28).CD4 + T cells have been described to contribute to anti-tumor immunity through a variety of mechanisms including direct killing of tumor cells, augmenting the tumor microenvironment through local secretion of effector cytokines, and providing help to CD8 + T cells (29)(30)(31)(32)(33). Our study complement prior reports that necroptotic signaling in tumors (34) and cardiac allografts (35) can bolster effector CD4 + T cell responses.Thus, different methods of ICD induction can elicit distinct mechanisms to confer anti-tumor immunity.
Consistent with the importance of type I IFN in anti-tumor immunity (24,36,37), type I IFN signaling was also critical for the protection conferred by necroptosis immunization.This type I IFN response likely originates from the necroptotic cells in a cell death-dependent manner, since the modest induction of IFNβ and ISGs was abrogated when cell death was inhibited by Mlkl inactivation.How might necroptosis promote this interferon response?Recent reports have shown that mitochondrial DNA (mtDNA) accumulates in the cytosol when necroptosis was induced in tumors in response to irradiation and Cisplatin (36, 37).The release of cytosolic mtDNA instigated tumor-intrinsic production of IFNβ and ISGs via the cGAS/STING pathway (36, 37).In this regard, it is noteworthy that MLKL can translocate to the nuclear and mitochondrial membranes during necroptosis (38,39).It is tempting to speculate that MLKLdependent pore formation may facilitate release of mtDNA to stimulate cGAS/STING, which in turn induces the rst wave of IFN within the necroptotic tumor cells.However, the precise role of MLKL in mediating these effects remains to be elucidated.Further, since type I IFN signaling in the host is also required for protection mediated by NEC immunization, our data support a model in which the initial wave of IFN signal continues to propagate in the host after clearance of dying necroptotic cells to achieve optimal anti-tumor effects.However, the cellular mediators of this host response remain an open question.Nevertheless, our data suggests that, cell-intrinsic type I interferon signaling plays a key role in promoting the immunologic consequences of necroptosis.

In vivo prophylactic dying tumor cell immunization
Tumor cells were seeded on 15-cm tissue culture dishes and cell death was induced in vitro by treating cells with DOX (1 µg/ml) for 9 hours followed by MG132 (4 µM, APExBio, Houston, TX, USA) for 4.5 hours.Dying tumor cells were then collected, washed twice in PBS (Thermo Fisher Scienti c, Waltham, MA, USA), then re-suspended at 7.5 x 10 6 cells/mL in PBS.Mice were immunized subcutaneously with 7.5 x 10 5 cells (100 µl) in the right ank.On day 8 after vaccination, mice were challenged subcutaneously on the left ank with 5 x 10 5 live tumor cells suspended in serum free-DMEM mixed 1:1 with Matrigel (Matrigel® Basement Membrane Matrix, LDEV-free, Corning Life Sciences, Tewksbury, MA, USA).Tumor growth on the challenge site was evaluated using calipers.Tumor volume was calculated using the formula: 0.5 x long axis x short axis 2 .Mice were euthanized if tumors exceeded 2000 mm 3 .
In vivo antibody administration For INFAR1 blockade, 1 mg of anti-IFNAR-1 antibody (clone MAR1-5A3, Bio X Cell, Lebanon, NH, USA) or isotype control was administered to mice intravenously via retroorbital injection the day prior to dying cell immunization.For T cell depletion, 350 µg of anti-CD8 (clone YTS 169.4,Bio X Cell), anti-CD4 (clone GK1.5, Bio X Cell), or Isotype control was administered to mice intravenously prior to dying cell immunization.Where indicated, an additional 150 µg of anti-CD8, anti-CD4, or Isotype control was administered on the day prior to dying cell immunization.

OT-II adoptive transfer
For OT-II adoptive transfer experiments, spleens were collected from congenic OT-II mice, mechanically homogenized and ltered through a 70 µM cell strainer.Erythrocytes were then lysed using ACK Lysis Buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA).Splenocytes were subsequently counted and the percentage of CD4 + T cells was determined by ow cytometry.Splenocytes were resuspended in RPMI at 10 x 10 6 CD4 + T cells/mL and 100 µl was administered to mice intravenously via retroorbital injection.
Cell death was induced in tumor cells with DOX (1 µg/ml) for 9 hours followed by MG132 (4 µM, APExBio) for 4.5 hours.Dying cells were collected for co-culture.Dying tumor cells and splenocytes were co-cultured at a 10:1 ratio in a 24 well plate with 0.5 mL of RPMI 1640 with 10% fetal bovine serum (FBS), 1% Non-essential Amino Acids, 1% sodium pyruvate, 2 mM L-glutamine, and 1% penicillin-streptomycin.Cells were harvested 8 and 24 hours later for ow cytometry analysis.

Flow cytometry
Single cell suspensions were obtained from tumors by digesting minced tumor tissue in complete RPMI containing type IV collagenase (1 mg/ml, Sigma C-5138) and deoxyribonuclease I (20 IU/ml, Sigma D4263-1VL) at 37°C with gentle agitation for 30 minutes followed by tissue homogenization.Cell suspension was then passed through a 70 µM cell strainer.Erythrocytes were lysed using ACK Lysis Buffer.Two million cells were stained with LIVE/DEAD xable aqua dead cell stain kit (Thermo Fisher Scienti c) for 30 mins at 4°C.Cells were incubated with Fc-blocking antibody (clone 2.4G2) for 15 mins prior to incubation with uorochrome-conjugated antibodies in 1x PBS, 2% FBS, and 2 mM EDTA at 4°C for 30 mins.Flow cytometry was performed on a BD Fortessa instrument.Analysis of ow cytometry data was done using FlowJo Treestar) software (version 10.8.1).

NanoString RNA analysis and qRT-PCR
To assess tumor cell cytokine production, cell death was induced in tumor cells using DOX and MG132.Total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen).For Nanostring analysis, RNA was run on a NanoString nCounter Pro Analysis System using an nCounter Mouse Tumor Signaling 360 Panel (Nanostring, Seattle, WA, USA).Data were normalized and analyzed using ROSALIND software (NanoString).We thank the Duke University School of Medicine for the use of the Microbiome Core Facility, which provided NanoString Gene Expression service.For qPCR, cDNA was synthesized using the iScripts cDNA synthesis kit (Bio-Rad 170-8891).Thermal cycling reaction was then performed using iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA 170-8882) and a CFX Connect Real-Time PCR Detection System (Bio-Rad).Cycle threshold (CT) values for target genes were normalized to CT values of the housekeeping gene Tbp1 (ΔCT = CT(Target) -CT(Tbp1)) and subsequently normalized to baseline control values (ΔΔCT = ΔCT(Experimental) -ΔCT(Control)).

RNAseq
Total RNA was extracted from single cell suspensions from tumor tissues.A mRNA library was prepared using the DNBSEQ platform by BGI with data ltering using the SOAPnuke software (42).HISAT2 was selected to map the ltered sequenced reads to the reference genome.BAM les containing mapping results were counted using the featureCounts function using Python.Counting was performed using the mouse genome for comparison.Downstream analyses were performed using iDEP.96web interface (43).DEG analysis was then performed using DESeq2 considering all genes with FDR ≤ 0.1 and 1 ≤ Log2FC ≤ -1.Functional analysis of genes with FDR ≤ 0.1, regardless of Log2FC, comprised of GO and GSEA (Gene Set Enrichment Analysis) analyses.For GSEA, gene sets used in this assessment included curated gene sets, known pathways (KEGG), and gene ontology terms (Biological Process & Molecular Function).

Incucyte Cell Death Assays
Cells were seeded in a 96-well plate with 10,000 cells per well in 200 µl complete growth medium.Eight hours prior to cell death initiation, medium was exchanged for Complete DMEM with DOX (1 µg/ml) or DMSO.Cells were subsequently treated with MG132 (4 µM, APExBio).Imaging was subsequently performed using the IncuCyte S3 (Sartorius, Göttingen, Germany; version 2021C).Nine images per well were captured, analyzed, and averaged.Cell death was assessed through measuring uptake of YoYo-1 (50 nM, Thermo Fisher Scienti c) and expressed as the area of YoYo-1 + cells as a percentage of the total phase area.In experiments where zVAD-fmk and GSK'843 were used, zVAD-fmk (20 µM, APExBio) was administered 30 minutes prior to treatment with GSK'843 (20 µM, Sigma) and MG132.

Western blot
Cell lysates were prepared in RIPA buffer containing 0.

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Figure 7
Figure 7 : Statistical analysis was performed in GraphPad Prism (version 9).Unpaired two-tailed Student's t-test was used to compare two independent groups.Tukey's multiple comparison test, or one-way Analysis of variance (ANOVA) or two-way ANOVA were used to compare multiple (> 2) groups with one or two independent variables, respectively; with multiple comparisons tests as indicated.P values > 0.05 were considered statistically non-signi cant.*p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001. Statistics